The mass spectrometric analysis of the major chain d1, one of the four heme-binding chains in earthworm hemeglobin of Lumbricus terrestris, revealed a difference between the measured and the sequence derived masses indicating errors in the reported sequence. The purpose of this study is to clarify the possible sequence ambiguity by mass spectrometric peptide mapping experiments. After the digestion of chain d with a number of enzymes, proteolytic peptides in the region of 51-140 were identified and indicated a correct sequence assignment in this region. In addition, digestion with V8 protease revealed an sequence error in position 58 (E instead of Q). N-terminal Edman sequencing allowed the correction of misidentified amino acids 7 (G7S), 23 (Q23E) and 33 (K33R). The refined sequence (1-140) results in a molecular mass of 15961 Da which is 28 Da below the measured mass of 15989Da. Current efforts are directed to resolve the remaining discrepancy.